Development of deep proteomic sequencing platforms for molecular markers in DLBCL Diffuse large B cell lymphoma (DLBCL) is a genetically and clinically heterogeneous disease that comprises the most common non-Hodgkin's lymphoma. To date efforts to unearth the molecular pathogenesis of DBLCL, identify therapeutic targets, and stratify patients according to specific sets of molecular lesions have relied on genome-level analyses that identified distinct substructure classifications and pathogenic mechanisms, including B-cell receptor (BCR)-dependent survival signaling. The potential biomarkers that have emerged from these studies await further validation. Quantitative proteomics has the power to reveal additional, novel molecular signatures at the protein/pathway level that may otherwise escape detection through genome-based approaches due to post-transcriptional and post-translational mechanisms. However, use of quantitative proteomics to profile molecular signatures and facilitate biomarker discovery in clinical tissues has been hampered by critical barriers such as poor detection of low-abundance proteins, overly complex sample workflows that are often incompatible with the protein quantities available in primary tumors, and low throughput. We have developed a novel and very high peak capacity liquid chromatography system coupled directly to mass spectrometry that can address these barriers and achieve extensive and quantitative proteome coverage. In the fullness of time, this platform will enable analyses of sufficiently large clinical cohorts to provde statistically-powered identification of protein biomarkers. In preliminary studies, we utilized a prototype version of our deep proteome sequencing platform to interrogate the proteome of primary DLBCL tumor biopsies and patient derived DLBCL cell lines. This analysis revealed previously unappreciated pathway-level heterogeneity in nutrient/fuel utilization and associated proliferation and survival advantages that segregate with the presence/absence of functional BCR signaling. In response to FOA PA-12-220, which encourages development of specific technologies for quantitative detection of novel biomarkers associated with hematopoietic malignancies, we propose to test, validate and improve our proteomics platform for systematic interrogation of both proteome (Aim 1) and phosphoproteome (Aim 2) signatures in primary DLBCL tumors. Our goal is to identify and validate coordinate pathways that are differentially enriched in molecular subsets of DLBCL. Our study plan will reveal putative new stratification markers for DLBCL and credential our deep protein sequencing LC-MS/MS platform for future use on much larger NHL patient cohorts.